STRUCTURAL CHARACTERIZATION OF AN ANALOG OF THE MAJOR RATE-DETERMINING DISULFIDE FOLDING INTERMEDIATE OF BOVINE PANCREATIC RIBONUCLEASE-A

The major rate-determining step in the oxidative regeneration of bovin e pancreatic ribonuclease A (RNase A) proceeds through des-[40-95] RNa se A, a three-disulfide intermediate lacking the Cys40-Cys95 disulfide bond. An analog of this intermediate, [C40A, C95A] RNase A, has been characterized in terms of regular backbone structure and thermodynamic stability at pH 4.6. Nearly complete backbone H-1, N-15, and C-13 res onances, and most C-13(beta) side-chain resonances have been assigned for the mutant RNase A using triple-resonance NMR data and a computer program, AUTOASSIGN, for automated analysis of resonance assignments. Comparisons of chemical shift data, (3)J(H-1(N)-H-1(alpha)) coupling c onstants, and NOE data for the mutant and wild-type proteins reveal th at the overall chain folds of the two proteins are very similar, with localized structural perturbations in the regions spatially adjacent t o the mutation sites in [C40A, C95A] RNase A. More significantly, H-1/ H-2 amide exchange and thermodynamic data reveal a global destabilizat ion of the mutant protein characterized by a significant difference in the midpoint of the thermal transition curves (Delta T-m of 21.8 degr ees C) and a significant increase in the slowest exchanging backbone a mide H-1/H-2 exchange rates (10(2)-10(6)-fold faster in the hydrophobi c core of [C40A, C95 A] RNase A). Comparisons of the entropy Delta S d egrees(T) and enthalpy Delta H degrees(T) unfolding between wild-type and [C40A, C95A] RNase A reveal that some of the global destabilizatio n of the mutant protein arises from entropic and enthalpic changes in the folded state. Implications of these observations for understanding the role of des-[40-95] in the folding pathway of RNase A are discuss ed.